Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed in tissues, particularly in blood vessels, suggesting a role for this enzyme for inactivating circulating methyleneamines (Lyles, Prog. Brain Res., 106:293-303, 1995). Many investigators have focused their efforts on finding an endogenous ligand for SSAO and on looking for inhibitors of this enzyme (Precious et al., Biochem. Pharmacol., 37:707-713, 1988; Crosbie and Callingham, J. Neural Transm. Suppl., 41:427-432, 1994; Elliot et al., Biochem. Pharmacol. 38:1507-1515, 1989; Boomsma et al., Comp. Biochem. Physiol. C Toxicol. Pharmacol., 126:69-78, 2000; Yraola et al., J. Med. Chem. 49:6197-6208; WO 02/066669). Others have searched for alternative physiological roles of SSAO. For example, SSAO, also called vascular adhesion protein-1 (VAP-1), has been shown to be up-regulated under inflammatory conditions and to mediate the binding of lymphocytes (WO 98/53049). The instant invention utilizes the enzyme activity of SSAO to measure the metabolic stability of test agents in cells and biological samples.
Metabolic stability of test agents has become a critical factor in drug development, thus, the instant invention solves the problem of identifying potential test agents that are subject to metabolism by SSAO. Advances in chemistry, molecular biology and high-throughput technology have provided drug discovery programs with the ability to screen an enormous number of compounds against a large number of targets to identify lead compounds. These leads then undergo more selection criteria to identify those compounds with optimal “drug-like” properties (i.e., adequate physico-chemical stability, solubility, safety, efficacy, in vivo disposition). Significant efforts are directed toward identifying and eliminating compounds (or compound classes) that are not likely to have “drug-like” properties at earlier stages of discovery. The three main reasons a drug fails during clinical trials are lack of efficacy, unacceptable adverse effects, and unfavorable ADME (absorption, distribution, metabolism, excretion) properties. Therefore the ultimate success of a compound is not only defined by its biological activity and potency, but also by its ADME/toxicity properties. As a result, lead optimization programs have incorporated screens to select drugs with desirable ADME/toxicity properties to enhance the likelihood that new lead compounds will have success in the clinic. Metabolic transformation of drug molecules represents a key process by which drugs are cleared from the body. Given the wide distribution of SSAO in many bodily compartments, we believe SSAO may be an important enzyme that contributes to a drug's potential metabolic liability. Thus, the instant invention uses methods of determining the metabolic stability of test agents exposed to SSAO. Methods directed to determining the metabolism of a drug or other test agent by enzymes involved in biotransformation, in particular SSAO, have important implications for drug development.
In in vitro studies, compound A (U.S. Pat. No. 6,977,263 B) was incubated with plasma of various species including human. Compound A was observed to be more stable in human plasma in comparison to sheep, guinea pig, rat, and mouse up to one hour. Additionally, no significant metabolism or degradation was observed for the compound in human plasma up to 4 hours with or without semicarbazide. However, pharmacokinetic data from the “first in man” study showed that the same compound was rapidly metabolized and to a far greater extent than that demonstrated in vitro using plasma from human and from other species. Subsequent studies confirmed that membrane-bound SSAO was primarily responsible for the metabolism of the compound in man. These data indicate that soluble SSAO found in human plasma does not possess the same level of enzyme activity and/or substrate specificity as the membrane bound SSAO under physiological conditions. Therefore, as illustrated by compound A, measuring human plasma stability is not predictive of SSAO stability in man and thus is of limited utility in selecting pharmaceutical agents as clinical candidates.